The androgen receptor (AR) is central to prostate cancer development, progression, and drug resistance. The AR is a nuclear transcription factor (TF) that binds to DNA and regulates gene activity. The set of genome-wide AR-DNA binding sites is termed the AR cistrome. A complex interplay between AR and its co-regulators determines the genes targeted for transcriptional regulation. Using newly developed techniques for AR chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) in human prostate specimens, we unambiguously show that the AR program is highly dynamic and is determined, at least in part, by the presence of available co-regulators. In particular, the pioneer TF FOXA1 and the prostate lineage- specific TF HOXB13 appear to co-localize at tumor tissue-specific AR sites. The overall objectives of the present proposal are to characterize the mechanisms underlying AR reprogramming during tumorigenesis and - for the first time - to characterize the AR program in human tissue during progression from localized prostate cancer to metastatic, drug-resistant disease. In the first aim, ChIP-seq will be performed in cell line models for AR-expressing normal prostate epithelium (LHSAR) and prostate cancer (LNCaP and VCaP). Changes in the cistromes of AR, FOXA1 and HOXB13 will be measured as each TF is knocked down via shRNA and genome editing or overexpressed via lentiviral transduction. RNA-seq will also be performed for each cell line condition to determine the genes affected by epigenetic reprogramming. In the second and third aims, the epigenetic landscape of the AR will be systematically charted via ChIP-seq in human specimens: localized tissue, untreated metastatic tissue, castration-resistant metastases and enzalutamide-resistant disease. Completion of the novel experiments outlined in this proposal will provide an unparalleled look at how master transcription factors drive prostate cancer progression. Specifically, we will discover: (i) how the AR is reprogrammed during prostate cancer development and progression, and which co-regulators are facilitating this process, (ii) how the AR is reprogrammed during the acquisition of resistance to enzalutamide and, if so, which co-regulators are facilitating this process, and (iii) other non-AR master regulators that are driving prostate cancer progression. The proposed experiments will also enable us to identify the target genes that are affected by AR reprogramming. The project will result in an atlas of the epigenetic landscape as the disease progresses to the castration-resistant metastatic state, which is uniformly fatal. The genetic loci and target genes comprising this dataset will stimulate new targets for therapeutic intervention.